Encoding hierarchical assembly pathways of proteins with DNA

Abstract

The structural and functional diversity of materials in nature depends on the controlled assembly of discrete building blocks into complex architectures via specific, multistep, hierarchical assembly pathways. Achieving similar complexity in synthetic materials through hierarchical assembly is challenging due to difficulties with defining multiple recognition areas on synthetic building blocks and controlling the sequence through which those recognition sites direct assembly. Here, we show that we can exploit the chemical anisotropy of proteins and the programmability of DNA ligands to deliberately control the hierarchical assembly of protein-DNA materials. Through DNA sequence design, we introduce orthogonal DNA interactions with disparate interaction strengths ("strong" and "weak") onto specific geometric regions of a model protein, stable protein 1 (Sp1). We show that the spatial encoding of DNA ligands leads to highly directional assembly via strong interactions and that, by design, the first stage of assembly increases the multivalency of weak DNA-DNA interactions that give rise to an emergent second stage of assembly. Furthermore, we demonstrate that judicious DNA design not only directs assembly along a given pathway but can also direct distinct structural outcomes from a single pathway. This combination of protein surface and DNA sequence design allows us to encode the structural and chemical information necessary into building blocks to program their multistep hierarchical assembly. Our findings represent a strategy for controlling the hierarchical assembly of proteins to realize a diverse set of protein-DNA materials by design.

Keywords: DNA; hierarchy; nanotechnology; protein assembly; supramolecular chemistry.

Scheme 1.

Design of Sp1m chemical surface and proposed hierarchical assembly schemes. (A) Native Sp1 (Left) presents multiple primary amines (lysines and N termini, blue) and no cysteines (red) on its surface. Three mutations were designed to remove two native lysines and introduce one cysteine per subunit. Due to the dodecameric structure of Sp1m, these mutations define the chemical anisotropy across the protein surface with amine residues only on the axial face and cysteines located only on the equatorial face. (B) Proposed assembly schemes for building blocks containing strong or weak surface interactions at their axial or equatorial positions. Strong interactions direct the first stage of assembly, leading to multivalency among weak interactions that direct the second stage of assembly.

Fig. 1.

Synthesis and characterization of Sp1m-DNA conjugates. (A) Sp1m (1) was modified with DNA in three steps: (i) cysteines were first modified with Linker 1 (C) through a thiol-maleimide Michael addition click reaction to give Sp1m-N₃ (2); (ii) primary amines were then modified with Linker 2 (C) to generate 3 through reaction with an NHS-activated ester; (iii) TCO- and DBCO-modified DNA were reacted with 3 in one pot to generate an Sp1m-DNA building block (4). (B) Negative-stain TEM of 1. (Scale bar, 50 nm.) (Lower) Comparison of a model of Sp1m with a magnified region from the TEM image. (C) Chemical structures of heterobifunctional Linkers 1 and 2. (D) MALDI-TOF MS confirming the consecutive addition of a single molecule of each linker to each subunit of 1. (E) Denaturing PAGE (Left to Right) protein ladder, unreacted Sp1m (1), and purified Sp1m-DNA conjugate (4). The presence of two bands of approximately equal intensity, at higher molecular weight compared to 1, correspond to a roughly equal mixture of protein subunits with one and two DNA strands.

Fig. 2.

Characterization of the assembly of Sp1m with strong axial (A_S/A′_S) interactions. (A) Scheme showing the donor-quenching FRET experiment. In a typical experiment, a pair of complementary Sp1m-DNA conjugates were functionalized with Cy3- or Cy5-modified axial DNA, respectively. When well separated, excitation of Cy3 results in fluorescence from Cy3 (filled red circle). However, when Cy3 and Cy5 are in close proximity, FRET from excited Cy3 to Cy5 quenches the fluorescence of Cy3 leading to reduced fluorescent signal (empty red circle). (B) Temperature-dependent association of Sp1m-A_SE_NC and Sp1m-A′_SE_NC represented as fraction assembled versus temperature, where the fluorescence intensities at 65 and 20 °C correspond to a fraction assembled of 0 and 1, respectively (details in SI Appendix). (C) Negative-stain and (D) cryogenic TEM micrographs of slow-cooled Sp1m-A_SE_NC and Sp1m-A′_SE_NC. (Scale bars, 150 nm.)

Fig. 3.

Characterization of the assembly of Sp1m with strong equatorial (E_S/E′_S) interactions. (A) Schematic of the donor-quenching FRET experiment. (B) Temperature-dependent association of Sp1m-E_S and Sp1m-E′_S represented by plot of fraction assembled versus temperature. (C) Negative-stain TEM micrograph of slow-cooled Sp1m-E_S and Sp1m-E′_S. (Scale bar, 150 nm.) (D) Liquid AFM micrograph of slow-cooled Sp1m-E_S and Sp1m-E′_S. White arrow denotes line used for height profile in E.

Fig. 4.

FRET-based characterization of temperature-dependent hierarchical assembly processes. (A–C) Hierarchical assembly mediated by strong axial (A_S/A′_S) interactions. (A) Scheme showing the hypothesized assembly outcomes for two pairs of A_S/A′_S building blocks: Sp1m-A_SE_W1 with Sp1m-A′_SE_W1, and Sp1m-A_SE_NC with Sp1m-A′_SE_NC. Temperature-dependent association of (B) Sp1m-A_SE_W1 and Sp1m-A′_SE_W1 and (C) Sp1m-A_SE_NC and Sp1m-A′_SE_NC represented by plots of fraction assembled versus temperature. Both pairs show the first stage of assembly mediated by A_S/A′_S interactions but only with E_W1 is a second stage of assembly observed. (D–F) Hierarchical assembly mediated by strong equatorial (E_S/E′_S) interactions. (D) Scheme showing hypothesized assembly outcomes for two pairs of E_S/E′_S building blocks: Sp1m-A_WE_S with Sp1m-A_WE′_S, and Sp1m-A_NCE_S with Sp1m-A_NCE′_S. Temperature-dependent association of (E) Sp1m-A_WE_S and Sp1m-A_WE′_S and (F) Sp1m-A_NCE_S and Sp1m-A_NCE′_S represented by plots of fraction assembled versus temperature. Both pairs show the first stage of assembly mediated by E_S/E′_S interactions but only with A_W is a second stage of assembly observed.

Fig. 5.

Characterization of assembly outcomes from axial-first, equatorial-second hierarchical assembly processes. (A) Scheme showing 1D protein chains displaying equatorial E_W1 DNA homogenously. (B) Negative-stain TEM micrograph of slow-cooled assembly of Sp1m-A_SE_W1 and Sp1m-A′_SE_W1. (C) Scheme showing 1D protein chains displaying alternating equatorial E_W1 and E_W2 DNA. (D) Negative-stain TEM micrograph of slow-cooled assembly of Sp1m-A_SE_W1 and Sp1m-A′_SE_W2. (Scale bars, 150 nm.)